1. Field of the Invention
The present invention relates to nucleic acid hybridization with crosslinkable oligonucleotide probes, and in particular, (1) a chemical synthesis route for the production of psoralen monoadducted oligonucleotide probes, (2) a method for the site-specific placement of a crosslinking site and/or a cross linking reagent during the enzymatic amplification of one or more segments of one or more nucleic acid targets, and (3) a hybridization method for discriminating between two or more nucleic acid base sequences in one or more nucleic acid targets that differ by a single base.
2. Description of the Prior Art
The ability of two polymers of nucleic acid containing complementary sequences to find each other and anneal through base pairing interactions is a well-recognized phenomenon. The initial observations of the "hybridization" process by Marmur and Lane, Proc. Nat. Acad. Sci., U.S.A. 46, 453 (1960) and Doty et al., Proc. Nat. Acad. Sci., U.S.A. 46, 461 (1960) have been followed by the refinement of this process into an essential tool of modern biology.
Initial hybridization studies, such as those performed by Hayashi et al., Proc. Nat. Acad. Sci., U.S.A. 50, 664 (1963), were performed in solution. Further development led to the immobilization of the target DNA or RNA on solid supports. With the discovery of specific restriction endonucleases by Smith and Wilcox, J. Mol. Biol. 51, 379 (1970), it became possible to isolate discrete fragments of DNA. Utilization of immobilization techniques, such as those described by Southern, J. Mol. Biol. 98, 503 (1975), in combination with restriction enzymes, has allowed for the identification by hybridization of single copy genes among a mass of fractionated, genomic DNA.
In spite of the progress made in methodology, a number of problems have prevented the wide scale use of hybridization as a tool in human diagnostics. Among the more formidable problems are a) the inefficiency of hybridization, b) the low concentration of specific target sequences in a mixture of genomic DNA, and c) the hybridization of only partially complementary probes and targets.